Propagation by somatic embryogenesis refers to methods whereby embryos are produced in vitro from small pieces of plant tissue or individual cells. The embryos are referred to as somatic because they are derived from the somatic (vegetative) tissue, rather than from the sexual process. Vegetative propagation via somatic embryogenesis has the capability to capture all genetic gain of highly desirable genotypes. Furthermore, these methods are readily amenable to automation and mechanization. These qualities endow somatic embryogenesis processes with the potential to produce large numbers of individual clones for reforestation purposes.
It was not until 1985 that somatic embryogenesis was discovered in conifers (Hakman et al. 1985) and the first viable plantlets were regenerated from conifer somatic embryos (Hakman and von Arnold 1985). Since 1985, conifer tissue culture workers throughout the world have pursued the development of somatic embryogenesis systems capable of regenerating plants. The goal of much of this work is to develop conifer somatic embryogenesis as an efficient propagation system for producing clonal planting stock en masse. Additionally, the embryogenic system interfaces very well with genetic engineering techniques for production of transgenic clonal planting stock of conifers.
The two most economically important conifer genera are Picea (spruce) and Pinus (pine). Those working in conifer somatic embryogenesis have found that there is a striking difference between Picea conifers and Pinus conifers as to the ease with which somatic embryogenesis can be induced and plants regenerated (Tautorus et al. 1991). Indeed, if one evaluates the success of somatic embryogenesis in conifers among species of these two important genera, it is clear that significantly more success has been achieved with initiating and establishing embryogenic cultures of Picea than with Pinus.
Among Picea species embryogenic culture initiation frequencies are relatively high; as high as 95% from immature zygotic embryos and as high as 55% from mature zygotic embryos harvested from fully developed, dry seeds (Tautorus et al. 1991). Researchers at the British Columbia Research Corporation have routinely reported initiation frequencies of about 27 percent in interior spruce (a mixture of Picea glauca and Picea englemannii). Moreover, these researchers have found this level of about 27 percent initiation frequency to be acceptable for the operational production of somatic embryo plants for field planting. Thus conifer somatic embryogenesis workers utilizing Picea species (and commercially important Douglas-fir) have been successful in developing culture initiation and maintenance systems that enable the routine production of plants from a range of families and genotypes (thereby not limiting the genetic material that is able to be deployed to the field).
In contrast, the progress achieved with somatic embryogenesis in Pinus species has been much less encouraging than that achieved with Picea species. The recalcitrance of Pinus species for initiation of embryogenic cultures is well documented. This is especially true for pines commonly found in the southeastern United States (known in the industry as Southern yellow pines). For example, initiation frequencies of about 1 to 5% are routinely cited by those working with Pinus species (Gupta and Durzan 1987, Becwar et al. 1988, Jain et al. 1989, Becwar et al. 1990). The single report claiming a 54% initiation rate from immature zygotic embryos of Pinus strobus (Finer et al. 1989) has yet to be repeated or duplicated by others working with this species (Michler et al. 1991).
Recently researchers working with Pinus species plants have achieved some important advances. In commonly assigned U.S. Pat. Nos. 5,413,930 and 5,506,136 (which are hereby incorporated by reference), Becwar et al. disclose multi-step methods that are able to complete the entire somatic embryogenesis regenerative process, from explant collection to planting, for historically recalcitrant Southern yellow pines (i.e., Pinus taeda, Pinus serotina, Pinus palustris, and Pinus elliottii), Pinus rigida, and hybrids thereof
In commonly assigned U.S. Pat. No. 5,491,090 (which is hereby incorporated by reference), Handley et al. taught a process that improved the above-noted methods by enabling the practitioner to replace the semi-solid maintenance culture media taught by Becwar et al. with liquid suspension culture media.
While the methods taught in U.S. Pat. Nos. 5,413,930, 5,506,136 and 5,491,090 have produced thousands of somatic embryos and hundreds of plants in the field, these embryos and plants have often been from a limited number of families or genotypes within a family. Therefore, even though these patented methods have achieved considerable success in both establishing embryogenic cultures of Pinus and in producing large numbers of field grown plants, these methods have proven to be somewhat limited by variable culture initiation frequencies experienced by different genetic families. Indeed, it is believed that the primary limiting factor in achieving clonal forestry in these pines has been the inability to produce embryogenic cultures from some of the very best genetic material and, subsequently, production of somatic embryo plants for field testing and eventual clonal deployment (Handley et al. 1995). Simply put, a major problem limiting commercial development of the above-noted methods is that they tend to exhibit relatively low initiation frequencies in certain explants due to the genetic specificity of those explants.
The present invention corrects this problem of relatively low cell culture initiation frequencies. Indeed, this improved method results in increases of from about 80% to 600% in the number of embryogenic culture initiations achieved when compared to the methods taught in U.S. Pat. Nos. 5,413,930, 5,506,136 and 5,491,090 (see Examples below). This improvement is highly significant because it ensures that more embryogenic cultures survive to the culture maintenance phase, thereby allowing more genotypes to be subsequently available for field testing and production of clonal planting stock.
Having a low initiation frequency can severely limit the potential applications of somatic embryogenesis in Pinus species for large-scale production of genetically improved conifers for the following reason. Skilled artisans in the conifer tissue culture field recognize that the use of embryogenic cultures derived from juvenile explants (e.g., zygotic embryos derived from seed) necessitate that the resulting regenerated plants be field tested prior to large-scale production. Only selected genotypes which show the potential for producing significant genetic gain in such field tests will subsequently be propagated by somatic embryogenesis. It will, therefore, be necessary to screen numerous genotypes from desirable parents, initiate embryogenic cultures, cryopreserve each genetically different culture, regenerate plants from each genetically different culture, field test plants from each genotype, and choose select genotypes for large scale production via somatic embryogenesis. Low culture initiation frequencies pose severe limitations for implementing this strategy. Indeed, an unbeknownst selection process may occur when low initiation frequencies exclude a majority of the genotypes. In the case of Pinus species where initiation frequencies can be very low (e.g., 1 to 5%) one could be selecting for embryogenic potential, but selecting against improved growth potential (which may be in the 95 to 99% of the genotypes eliminated as non-embryogenic).
As noted above, another major problem plaguing current somatic embryogenesis methods is that it has been extremely difficult to establish sufficient numbers of embryogenic cultures from some of the best genetic families of loblolly pine. A series of experiments have shown that a large percentage of those explants displaying commercially acceptable initiation frequencies when propagated via the above-noted patented methods have proven to be derived from families of average or poor genetic potential.
The present method corrects this problem by allowing one to produce large numbers of embryogenic cultures from a wide range of genetic materials, including materials of very high genetic value.
Somatic embryogenesis processes utilized with conifers (particularly the Pinus species) usually involve seven general steps: 1) culture initiation, 2) culture maintenance, 3) embryo development, 4) embryo maturation, 5) embryo germination, 6) conversion, and 7) plant growth (field planting). The culture media employed in the different steps are key components of effective somatic embryogenesis regeneration systems.
U.S. Pat. Nos. 5,413,930 and 5,506,136 teach the use of semi-solid culture media during the culture initiation and the culture maintenance steps. These culture media are generally composed of six groups of ingredients: inorganic nutrients, vitamins, organic supplements, a carbohydrate source, phytohormones, and a gelling agent. U.S. Pat. No. 5,491,090 teaches the replacement of the semi-solid maintenance culture media with liquid suspension culture media. This liquid suspension culture medium is generally composed of six groups of ingredients: inorganic nutrients, vitamins, organic supplements, a carbohydrate source, phytohormones, and activated carbon. The phytohormones used in conifer embryogenic systems have traditionally been an auxin and a cytokinin for the culture initiation and maintenance steps and abscisic acid for embryo development.
The improvement of the present method over the prior art (including the above-noted patented methods) lies in the fact that the present method adds abscisic acid to the earliest stage of the conifer somatic embryogenesis process--the initiation media. The above-noted patented methods do not contain abscisic acid in their initiation media. Indeed, the art's literature has traditionally taught away from using abscisic acid for the purpose of initiation, as heretofore it has been commonly believed by those skilled in the art that the only phytohormones necessary to initiate and maintain somatic embryogenesis were auxins and cytokinins.
In conifer somatic embryogenesis processes, abscisic acid (ABA) has traditionally been utilized after the culture initiation step--that is, in the later process steps concerning embryo development. The importance of ABA for these subsequent stages of development in zygotic embryogenesis is well known, and ABA has been used routinely to stimulate embryo development in somatic embryogenic systems (von Arnold and Hakman, 1988).
For example, U.S. Pat. No. 4,957,866 teaches the use of ABA in the embryo development media. Likewise, in U.S. Pat. Nos. 5,034,326 and 5,036,007 the growth regulator ABA along with activated carbon has been reported to be beneficial in the semi-solid development media for various conifers. U.S. Pat. No. 5,294,549 teaches the incorporation of ABA and gibberellic acid into maintenance media and late stage proembryo development media. In U.S. Pat. Nos. 5,187,092, 5,183,757, and 5,236,841 ABA is also used in the development step in conifer somatic embryogenesis. It is important to note that in all of these methods ABA is added to facilitate embryo development. The present method fundamentally differs from the prior art by utilizing ABA much earlier in the somatic embryo process (i.e., during the culture initiation step) to facilitate culture initiation.
ABA, which has been recognized as playing an important role in the later stages of conifer embryo development, has traditionally been employed concentrations of 0.1 to 15.0 mg/l (Gupta et al., 1991). It is commonly believed that cleavage polyembryony must be inhibited through the removal of auxins and cytolinins and the addition of ABA. However, the present invention shows that the addition of ABA to the pine embryogenic system seems to stimulate polyembryony at the early stages of culture initiation (see Examples).
Therefore, an object of the present invention is to provide an improved method for establishing embryogenic cultures for use in somatic embryogenesis processes for plants of the genus Pinus and Pinus interspecies hybrids.
Another object of the present invention is to provide an improved method for the regeneration of coniferous plants by somatic embryogenesis from these cultures.
A further object of the present invention is to provide an improved method for the establishment of embryogenic cultures from plants of the genus Pinus and Pinus interspecies hybrids so that these cultures can be further induced to regenerate stage 3 embryos when placed in the development stage, and further germinated and converted to yield viable plants for field planting.